A filter microfluidic chip for isolating circulating tumor cells

Abstract

The application discloses a filtering micro-fluidic chip for separating circulating tumor cells, and belongs to the technical field of high polymer materials, which comprises a cover plate and a bottom plate, the cover plate and the bottom plate are in a separable structure, and the opposite sides of the cover plate and the bottom plate are bonded with PDMS, and the PDMS is provided with a porous filter membrane area; a passage is arranged between the cover plate and the bottom plate, and the passage is communicated with the porous filter membrane area; the porous filter membrane area is configured with a porous filter membrane or a modified porous filter membrane, and the modified porous filter membrane is a composite membrane with a three-layer structure; the composite membrane comprises a base film layer, a membrane intermediate layer and an amphoteric ion polymer layer which are sequentially arranged from inside to outside, and the amphoteric ion polymer layer is provided with a pyridine group. The application can greatly improve the capture efficiency of tumor cells and improve the anti-biological pollution performance of the filter membrane.

Description

Technical Field

[0001] This invention belongs to the field of polymer materials technology and is a technology that combines a polymer porous filter membrane with a microfluidic chip, specifically involving a filter-type microfluidic chip for separating circulating tumor cells. Background Technology

[0002] Circulating tumor cells (CTCs) are malignant tumor cells that detach from the primary tumor or metastatic lesions and enter the bloodstream; they are considered essential for tumor metastasis. Clinical studies have shown that patient survival is inversely proportional to the number of CTCs (less than 5 CTCs per milliliter of blood sample in cancer patients with high survival rates), and that reducing or eliminating CTCs through primary or adjuvant therapy can prolong cancer patient survival. Therefore, the isolation and analysis of CTCs can provide information about tumor metastasis, tumor response after treatment intervention, and even guide personalized treatment for cancer patients.

[0003] Current methods for separating CTCs include the following:

[0004] Density gradient centrifugation: There are two methods, Ficoll and OncoQuick Plus. Since density gradient centrifugation may result in a high loss rate of CTCs, it is now generally only used for preprocessing of other enrichment methods.

[0005] Antibody-dependent isolation (ADC): This method is mainly divided into positive enrichment and negative enrichment. The principle is to use epithelial cell adhesion factor (EpCAM) and cytokeratin (CK) as detection markers. Antibody magnetic beads / nanoparticles are used to couple the epithelial cell surface marker EpCAM to tumor cell surface antigens. Fluorescently labeled antibodies are then used, followed by nuclear staining to identify tumor cells (CTCs). However, positive enrichment methods have many limitations. Not all tumor cells express EpCAM, and cell surface markers may be absent in cell lines, leading to low enrichment rates. Negative enrichment is a method that does not depend on CTC surface markers. During enrichment, other non-tumor cells in whole blood are first removed, and then tumor cells are enriched. Commonly used gradient methods, such as the Ficoll method, first remove red blood cells, then use CD45 labeling to remove white blood cells, and finally verify CTCs using immunochemical analysis or reverse transcription-polymerase chain reaction (RT-PCR). Similarly, negative enrichment also has many limitations. It cannot identify all abnormal cells enriched, and excessive white blood cells can interfere with CTC identification.

[0006] Based on cell size separation method: 1. Inertial microfluidic technology: when the fluid flows in a linear microchannel in a laminar flow, the suspended cells in the fluid are affected by the shear gradient lift and the wall effect lift, and under the action of the two forces, cells of different sizes produce different flow characteristics. By using different structures inside the chip, the separation of CTCs can be realized. But there is the disadvantage of low flux. 2. Deterministic lateral displacement (DLD) technology: when the fluid flow direction is at a certain angle with the microcolumn array in the chip, cells of different sizes have different movement trajectories during flow. Large size cells will be moved to one side, and small size cells will move along the original trajectory, thereby realizing the separation of CTCs. Size and deformability are common separation methods in microfluidic cell separation. 3. Membrane filtration technology: the diameter of most epithelial CTCs is 14-26 µm, while the diameter of WBCs is only 8-20 µm. By integrating microfluidic chips with various microfilters, when the sample flows through the microfluidic chip, due to the larger diameter of CTCs, they are trapped on the microfilter, while blood cells flow out with the buffer. In addition, compared with CTCs, WBCs are more likely to deform. Therefore, the larger size white blood cells trapped on the microfilter can be washed away by increasing the flow rate, while the CTCs with smaller deformation than white blood cells are still trapped on the microfilter.

[0007] The existing method based on density gradient centrifugation can separate part of CTCs, but the content of CTCs in blood is extremely small, and the purity of CTCs separated by this method is low; the separation method relying on antibodies can separate CTCs with high purity, but the cost is high and the time is long; inertial microfluidic technology and deterministic lateral displacement (DLD) technology can separate CTCs, but there is the problem of low flux, and the capture efficiency of CTCs is low, and the filter membrane is easy to be contaminated and blocked, and the cost is high. SUMMARY

[0008] The purpose of the present application is to provide a filter type microfluidic chip for separating circulating tumor cells, which can greatly improve the capture efficiency of tumor cells and improve the anti-biocontamination performance of the filter membrane.

[0009] The technical scheme adopted by the present application to achieve the above purpose is:

[0010] A filter type microfluidic chip for separating circulating tumor cells,

[0011] The cover plate and the bottom plate are separable, and the opposite sides of the cover plate and the bottom plate are bonded with PDMS, and the PDMS is provided with a porous filter membrane area; a passage is arranged between the cover plate and the bottom plate, and the passage is communicated with the porous filter membrane area; the porous filter membrane area is configured with a porous filter membrane or a modified porous filter membrane, and the modified porous filter membrane is a composite membrane with a three-layer structure; the composite membrane II comprises a base film layer, a membrane intermediate layer and a zwitterionic polymer layer arranged in sequence from inside to outside, and the zwitterionic polymer layer has a pyridine group.

[0012] In the preparation process of the modified porous filter membrane, the surface of the base film is compounded with the membrane intermediate layer and the zwitterionic polymer layer, so that the hydrophilicity of the filter membrane is enhanced, so that when the sample is injected, the surface of the filter membrane is first combined with the water in the blood sample to form a water film, so that the adhesion of the biological macromolecules such as protein molecules and antibiotics in the blood sample to the surface of the filter membrane can be reduced, so that the pollution of the blood sample to the filter membrane can be reduced, and the growth of microorganisms can be prevented, that is, the anti-biological pollution performance of the filter membrane is improved, and the plugging probability of the filter membrane is also reduced.

[0013] Preferably, the pore size of the modified porous filter membrane in the filter type microfluidic chip is 5-15 μm.

[0014] Preferably, the pyridine group in the zwitterionic polymer layer comes from 2-vinylpyridine and / or 4-butene pyridine.

[0015] According to one aspect of the present application, a modified porous filter membrane is provided, which is a composite membrane with a three-layer structure; comprising a base film layer, a membrane intermediate layer and a zwitterionic polymer layer arranged in sequence from inside to outside, and the zwitterionic polymer layer has a pyridine group, and the pyridine group comes from 2-vinylpyridine and / or 4-butene pyridine.

[0016] The surface of the modified porous filter membrane is a zwitterionic polymer layer, and has a pyridine group, which helps to enhance the hydrophilicity, so as to reduce the adhesion of biological macromolecules in the blood sample to the surface of the filter membrane, so as to reduce the pollution of the filter membrane, that is, to improve the anti-biological pollution performance of the filter membrane, and to reduce the plugging probability of the filter membrane. And due to the enhancement of the hydrophilicity of the modified porous filter membrane, the adhesion of tumor cells to the filter membrane is low, easy to separate, and the damage to the cells is small. On the other hand, the presence of amino and sulfonic acid groups helps to improve the uniformity of the water film combined on the surface of the composite membrane II; and the presence of amino and sulfonic acid groups helps to extend the side chain of the zwitterionic polymer, which plays a positive role in the process of intercepting tumor cells by the filter membrane.

[0017] According to one aspect of the present application, a preparation method of a modified porous filter membrane is provided, comprising the following steps:

[0018] S1. Preparation of the membrane intermediate layer: immerse the clean and dry base film in the polyethyleneimine mixed solution, react for 6-24 h, and obtain the composite film I with the membrane intermediate layer;

[0019] S2. Preparation of the grafted modified microfiltration membrane: treat the composite film I in S1 with the redox initiator, and react with the zwitterionic polymer monomer and 2-vinylpyridine and 4-butene pyridine, to obtain the base film with the grafted modified microfiltration membrane, denoted as the composite film II.

[0020] Preferably, in step S1, the polyethyleneimine mixed solution is configured by polyethyleneimine and Tris-HCl buffer solution, and further contains 3-phthalimido propyl aldehyde; the mass ratio of polyethyleneimine to 3-phthalimido propyl aldehyde is 1:0.5-2. The Tris-HCl buffer solution has a pH of 8.5 and a concentration of 50 mM; the mass-volume ratio of polyethyleneimine, 3-phthalimido propyl aldehyde and Tris-HCl buffer solution is 1 g:0.5-2 g:400-600 mL.

[0021] Preferably, in step S1, after the base film is immersed in the polyethyleneimine mixed solution, the reaction is carried out in a constant-temperature shaker at 20-35 ℃ and 120 r / min for 6-24 h.

[0022] Preferably, in step S2, the redox initiator is ammonium persulfate and sodium metabisulfite; and the mass concentration of ammonium persulfate and sodium metabisulfite is 1-5 mg / mL.

[0023] Preferably, in step S2, the redox initiator is dibenzoyl peroxide (BPO) and N,N-dimethyl benzene (DMA); and the mass concentration of DMA is 3-8 mg / mL, and the mass concentration of BPO is 15-30 mg / mL.

[0024] Preferably, in step S2, the zwitterionic polymer monomer is added before 2-vinylpyridine and 4-butene pyridine; the zwitterionic polymer monomer includes at least one of allyl trimethyl ammonium chloride, 2-acrylamido-2-methyl propane sulfonic acid and poly (methacrylic acid carboxy betaine).

[0025] Further, before 2-vinylpyridine and 4-butene pyridine are added, the mass concentration of the zwitterionic polymer monomer is 3-10 mg / mL.

[0026] Further, in step S2, the zwitterionic polymer monomer includes allyl trimethyl ammonium chloride and 2-acrylamido-2-methyl propane sulfonic acid; and before 2-vinylpyridine and 4-butene pyridine are added, the mass concentration of allyl trimethyl ammonium chloride and 2-acrylamido-2-methyl propane sulfonic acid is 1.5-5 mg / mL.

[0027] Preferably, in step S2, the volume ratio of 2-vinylpyridine and 4-butene pyridine is 1-2:1-3.

[0028] Further, the mass volume ratio of the zwitterionic polymer monomer, 2-vinylpyridine and 4-butene pyridine is 3-10 mg:0.5-2.5 mL:0.5-2.5 mL.

[0029] Preferably, in step S2, the composite film I is added with water and then added with the redox initiator, the treatment temperature is 35-50℃, and the treatment time is 10-30 min.

[0030] Preferably, in step S2, after the zwitterionic polymer monomer and 2-vinylpyridine and 4-butene pyridine are added, the reaction temperature is 35-50℃, and the reaction time is 1-3 h.

[0031] Preferably, in the preparation of the modified porous filter membrane, in the process of preparing the grafted modified microfiltration membrane in step S2, 2-vinylpyridine and 4-butene pyridine are added at the same time as butyl acrylate.

[0032] Preferably, the volume ratio of 2-vinylpyridine, 4-butene pyridine and butyl acrylate is 1-2:1-3:0.5-2.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] 1. In the preparation process of the modified porous filter membrane, the surface of the composite film I is grafted with a zwitterionic polymer, so that the hydrophilicity of the filter membrane is enhanced, so that when the sample is introduced, the surface of the filter membrane is first combined with water in the blood sample to form a water film, in this way, the adhesion of biological macromolecules such as protein molecules and antibiotics in the blood sample to the surface of the filter membrane can be reduced, so that the pollution of the blood sample to the filter membrane can be reduced, and the growth of microorganisms can be prevented, that is, the anti-biological pollution performance of the filter membrane is improved, and the probability of clogging of the filter membrane can also be reduced.

[0035] 2. The addition of 2-vinylpyridine and 4-butene pyridine in the preparation of the composite membrane II can greatly improve the capture efficiency of tumor cells. This may be because the addition of 2-vinylpyridine and 4-butene pyridine introduces amino and sulfonic acid groups in the synthesis of the composite membrane II, which helps to improve the uniformity of the water film combined on the surface of the composite membrane II, and these groups can also react with related groups on the surface of tumor cells, thereby enhancing the capture effect; in addition, the amino and sulfonic acid groups in 2-vinylpyridine and 4-butene pyridine may react with the zwitterionic allyl trimethyl ammonium chloride and 2-acrylamido-2-methylpropane sulfonic acid, thereby extending the side chain of the grafted zwitterionic polymer on the surface of the composite membrane I, further improving the structure of the modified composite membrane II, which plays a positive role in the process of tumor cell interception by the filter membrane.

[0036] 3. In the process of separating and enriching tumor cells by using the microfluidic chip assembled with the modified porous filter membrane, the adhesion of tumor cells to the filter membrane is low, which is easy to separate, and the damage to the cells is small. This may be because in the preparation of the composite membrane II, allyl trimethyl ammonium chloride and 2-acrylamido-2-methylpropane sulfonic acid groups are introduced on the surface of the base film, the hydrophilicity of the filter membrane is enhanced, so that the tumor cells are more easily detached during the reverse flushing process, and the damage is small. In addition, the addition of 2-vinylpyridine and 4-butene pyridine introduces pyridine groups in the synthesis of the composite membrane II, which may help to further reduce the force of the positive and negative atoms in the zwitterion on the surface of the cells. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The infrared spectrum of the composite membrane II obtained according to Example 1 of the present application and PET. DETAILED DESCRIPTION

[0038] The technical solutions of the present application will be further described in detail below in combination with specific embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0039] Example 1

[0040] Preparation of modified porous filter membrane

[0041] 1. Preparation of membrane intermediate layer

[0042] The base film was soaked in acetone for 12 h to remove impurities adsorbed on its surface, and then dried in a vacuum drying oven at 40°C for 1 h. The base film is a membrane chip from a microporous filter purchased from Hangzhou Baimai Medical Technology Co., Ltd., which is made of polycarbonate high molecular material, the thickness of the membrane chip is 11 μm ± 1 μm, and the micropore size is 10 μm.

[0043] Polyethyleneimine (PEI) is added to a Tris-HCl buffer solution (pH = 8.5, 50 mM), stirred uniformly, and then 3-phthalimido propionaldehyde is added, wherein the mass-volume ratio of PEI, 3-phthalimido propionaldehyde, and the Tris-HCl buffer solution is 1 g:1 g:500 mL.

[0044] The dried base film is completely immersed in the above-mentioned mixed solution of PEI, and is reacted in a constant-temperature shaking table at 25°C at 120 r / min for 12 h to form a film intermediate layer on the surface of the base film, which is denoted as composite film I; after the composite film I is washed with water for 3 times, water is added in a mass-volume ratio of 10 g:500 mL, and is oscillated in a constant-temperature shaking table at 25°C at 150 r / min for 30 min to remove unstable PEI and 3-phthalimido propionaldehyde. After the oscillation is completed, the obtained composite film I is dried in a vacuum drying box at 40°C for 1 h for standby use.

[0045] 2. Preparation of a graft-modified microfiltration membrane

[0046] The above-mentioned dried composite film I is added into water, and then N,N-dimethyl benzene (DMA) and benzoyl peroxide (BPO) are added as an oxidation-reduction initiator under an argon atmosphere, and the system is heated at 40°C in an oil bath for 20 min. The mass concentration of DMA is 5 mg / mL, and the mass concentration of BPO is 20 mg / mL. Then, the zwitterionic polymer monomer allyl trimethyl ammonium chloride and 2-acrylamido-2-methylpropanesulfonic acid are added into the reaction system, and the mass concentration of each of the two is 3 mg / mL. The above-mentioned system is heated at 40°C in an oil bath for 2 h to obtain a base film with a graft-modified microfiltration membrane, which is denoted as composite film II.

[0047] After the composite film II is washed with water for 3 times, water is added in a mass-volume ratio of 10 g:500 mL, and is oscillated in a constant-temperature shaking table at 25°C at 150 r / min for 30 min. After the oscillation is completed, the obtained composite film II is dried in a vacuum drying box at 40°C for 1 h to obtain a usable modified porous filtration membrane. The obtained modified porous filtration membrane composite film II is a composite membrane with a three-layer structure, which comprises, from inside to outside, a base film layer, a film intermediate layer, and a zwitterionic polymer layer.

[0048] The base film and the obtained composite film II are characterized by using a Fourier infrared spectrometer. The sample is prepared by using a potassium bromide tabletting method, the scanning wavelength range is 500 cm -1 ~4000 cm -1 ; the scanning line number is 32; the resolution is 4 cm -1 ; the obtained infrared spectrum is shown in Figure 1 . As can be seen from the figure, the composite film II has a peak at 3300 cm-1 3400cm -1 nearby appears strong and wide absorption peak, which belongs to N-H absorption peak; 1750cm -1 nearby absorption peak is still reserved, which is C=O absorption peak; in 1040cm -1 nearby absorption peak changes, which should be attributed to the action of sulfonate in zwitterion; in 1040cm -1 1260cm -1 nearby absorption peak changes, which may be attributed to the action of sulfonate. In 1400cm -1 nearby absorption peak changes, which may be attributed to the action of C-N bond. The above can verify that the reaction forms composite film II on the surface of composite film I.

[0049] Assemble filterable microfluidic chip

[0050] Adopt 1mm thick glass as bottom plate, 2mm thick glass as cover plate, and plasma permanently bond the bottom plate and cover plate with 500μm PDMS, and connect the cover plate and the bottom plate, and contain the PDMS. The two layers of PDMS in the middle correspond to the porous filter membrane area, and the modified porous filter membrane composite film II is arranged therein, and the shape of the composite film II is adapted to the porous filter membrane area, forming the main structure of the filterable microfluidic chip.

[0051] Among them, the cover plate sets the upper layer flow channel of the chip, the inlet and the outlet, and the inlet communicates with the upper layer flow channel of the chip; the lower glass cover plate sets the lower layer flow channel of the chip, and one end of the lower layer flow channel communicates with the outlet; and the end of the upper layer flow channel away from the inlet communicates with the porous filter membrane area, and the end of the lower layer flow channel away from the outlet also communicates with the porous filter membrane area. In this way, the inlet-upper layer flow channel-porous filter membrane area-lower layer flow channel-outlet forms a channel, and under the action of the external pump body, cell suspension and the like can flow therein, and the modified porous filter membrane can be used for separating tumor cells and the like therein.

[0052] Further, the filterable microfluidic chip is 55mm long and 25mm wide in whole. The cover plate and the bottom plate bonded with PDMS are set as an open structure, and the two can be separated, so as to facilitate the replacement of the filter membrane.

[0053] Further, the modified porous filter membrane is circular, and its radius is slightly larger than the radius of the porous filter membrane area, so as to prevent the cell suspension from leaking out.

[0054] In this embodiment, the filter hole diameter of the selected base film is 10μm, which is modified, and the obtained modified microporous filter membrane is used to assemble the filterable microfluidic chip. In addition, according to the needs, the filter hole diameter of the selected base film can also be 6μm, 10μm, 15μm and other specifications.

[0055] Example 2

[0056] The difference between this embodiment and embodiment 1 is that, in the preparation of the modified porous filter membrane, 2-vinylpyridine and 4-butene pyridine are added after the addition of the zwitterionic polymer monomer in the preparation of the graft-modified microfiltration membrane, and the volume ratio of 2-vinylpyridine and 4-butene pyridine is 1:1; and the mass-volume ratio of allyl trimethyl ammonium chloride, 2-acrylamido-2-methylpropane sulfonic acid, 2-vinylpyridine and 4-butene pyridine is 3mg:3mg:1mL:1mL.

[0057] Example 3

[0058] The difference between this embodiment and embodiment 1 is that, in the preparation of the modified porous filter membrane, 2-vinylpyridine and 4-butene pyridine are added after the addition of the zwitterionic polymer monomer in the preparation of the graft-modified microfiltration membrane, and the volume ratio of 2-vinylpyridine and 4-butene pyridine is 1:1; and the mass-volume ratio of allyl trimethyl ammonium chloride, 2-acrylamido-2-methylpropane sulfonic acid, 2-vinylpyridine and 4-butene pyridine is 3mg:3mg:1.5mL:1.5mL.

[0059] The other conditions and steps are the same.

[0060] Example 4

[0061] The difference between this embodiment and embodiment 1 is that, in the preparation of the modified porous filter membrane, 2-vinylpyridine and 4-butene pyridine are added after the addition of the zwitterionic polymer monomer in the preparation of the graft-modified microfiltration membrane, and the volume ratio of 2-vinylpyridine and 4-butene pyridine is 1:1; and the mass-volume ratio of allyl trimethyl ammonium chloride, 2-acrylamido-2-methylpropane sulfonic acid, 2-vinylpyridine and 4-butene pyridine is 3mg:3mg:2mL:2mL.

[0062] The other conditions and steps are the same.

[0063] Example 5

[0064] The difference between this embodiment and embodiment 1 is that, in the preparation of the modified porous filter membrane, 2-vinylpyridine, 4-butene pyridine and butyl acrylate are added at the same time when adding 2-vinylpyridine and 4-butene pyridine in the reaction system; and the volume ratio of 2-vinylpyridine, 4-butene pyridine and butyl acrylate is 1:1:0.5.

[0065] The other conditions and steps are the same.

[0066] Example 6

[0067] The embodiment differs from example 1 in that, in the preparation of the modified porous filter membrane, butyl acrylate is added when 2-vinylpyridine and 4-butene pyridine are added in the preparation of the graft-modified microfiltration membrane; the volume ratio of 2-vinylpyridine, 4-butene pyridine and butyl acrylate is 1:1:1.

[0068] The other conditions and steps are the same.

[0069] Comparative example 1

[0070] The comparative example differs from example 1 in that the base membrane is directly used to assemble the filter-type microfluidic chip.

[0071] Comparative example 2

[0072] The comparative example differs from example 2 in that, in the preparation of the modified porous filter membrane, 4-butene pyridine is replaced by an equal amount of 2-vinylpyridine in the preparation of the graft-modified microfiltration membrane.

[0073] The other conditions and steps are the same.

[0074] Comparative example 3

[0075] The comparative example differs from example 2 in that, in the preparation of the modified porous filter membrane, 2-vinylpyridine is replaced by an equal amount of 4-butene pyridine in the preparation of the graft-modified microfiltration membrane.

[0076] The other conditions and steps are the same.

[0077] Test example

[0078] 1. Capture efficiency of filter-type microfluidic chip on different tumor cells

[0079] Breast cancer MDA-MB-231 cells, lung cancer A549 cells and liver cancer HepG2 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum in a 37°C, 5% CO2 saturated humidity incubator.

[0080] Fresh peripheral blood of healthy volunteers was collected, and breast cancer MDA-MB-231 cells, lung cancer A549 cells and liver cancer HepG2 cells were added to the blood, respectively, so that the final concentration of tumor cells in the circulating liquid was 10 3 / mL, and after shaking and mixing, each filter-type microfluidic chip assembled in examples 1-6 and comparative examples 1-3 was used for filtration separation. When the cell liquid was filtered and separated, an external syringe pump was used to inject 3 mL of blood sample containing tumor cells into the filter-type microfluidic chip at a flow rate of 0.1 mL / min. After the blood sample was completely filtered, the syringe pump was turned off. Each blood sample was subjected to 10 parallel tests when each filter-type microfluidic chip was used for filtration separation.

[0081] After filtration, 5 parallel tests were randomly selected from each filter microfluidic chip, and the filter membrane was washed with 2% Triton X-100 buffer for 10 min, and then blocked with PBS buffer containing 10% FBS at room temperature for 1 h. PancytoKeratin Alexa 488 antibody and anti-CD45 antibody (PerCP / Cy5.5) were added and incubated in a wet box for 1 h, and then PBS buffer was used for hydration for 5 min. Then, DAPI fluorescent staining was added for 10 min, and anti-quenching mounting agent was added for mounting, and then observed and counted under a fluorescence microscope to calculate the capture efficiency. The remaining 5 parallel tests in each filter microfluidic chip were rotated by 180º, 0.5 mL of PBS was added to the inlet of the microfluidic chip for reverse flushing, and a basic membrane with a pore size of 6 μm was used to collect the tumor cells captured on each filter membrane, and fluorescent staining and counting were performed according to the above method to calculate the cell loss rate, and the cell loss rate = reverse flushing collected tumor cell number / total number of tumor cells captured on the filter membrane x 100%.

[0082] The capture efficiency of the filter microfluidic chip obtained in each example and comparative example for different tumor cells was calculated, and the results are shown in Table 1, and the cell loss rate was calculated, and the results are shown in Table 2.

[0083] Table 1 Capture efficiency of filter microfluidic chip for different tumor cells (%)

[0084]

[0085] Referring to the data in Table 1, compared with Comparative Example 1, the capture efficiency of the microfluidic chip obtained in Examples 1-6 and Comparative Examples 2-3 for different tumor cells was improved.

[0086] The capture efficiency of the microfluidic chip obtained in Example 1-4 for MDA-MB-231 cells is increased by 5.17%, 11.02%, 11.97%, and 13.06% respectively relative to Comparative Example 1; the capture efficiency of the microfluidic chip obtained in Example 1-4 for A549 cells is increased by 5.46%, 11.34%, 12.46%, and 13.30% respectively relative to Comparative Example 1; and the capture efficiency of the microfluidic chip obtained in Example 1-4 for HepG2 cells is increased by 5.96%, 10.35%, 11.20%, and 13.90% respectively relative to Comparative Example 1. It can be seen that, relative to the base membrane, the microfluidic chip assembled using the modified porous filter membrane can greatly improve the capture efficiency for different tumor cells, that is, the modified porous filter membrane has a better enrichment effect on tumor cells. That is, in the preparation process of the modified porous filter membrane, by grafting the zwitterionic polymer on the surface of the composite membrane I, the adhesion force of the biological macromolecules such as protein molecules and antibiotics in the blood sample to the surface of the filter membrane can be reduced, and the modified composite membrane II has an increased hydrophilic property, thereby enhancing the interception effect on tumor cells. This may be because, in the preparation of the composite membrane II, allyl trimethyl ammonium chloride and 2-acrylamido-2-methylpropanesulfonic acid groups are introduced on the surface of the base membrane, which is helpful for the adsorption of tumor cells, and especially the presence of the methyl ammonium chloride group and the sulfonic acid group helps to react with the related groups on the surface of the tumor cells, thereby enhancing the capture effect.

[0087] In addition, relative to Example 1, the capture efficiency of the microfluidic chip obtained in Examples 2-3 for the above three kinds of tumor cells is significantly improved, which shows that the addition of 2-vinylpyridine and 4-butene pyridine in the preparation of the composite membrane II can greatly improve the capture efficiency for tumor cells.

[0088] In addition, the capture efficiency of the microfluidic chip obtained in Comparative Examples 2-3 for MDA-MB-231 cells, A549 cells, and HepG2 cells is better than that of Comparative Example 1, but worse than that of Example 2, which shows that the simultaneous addition of 2-vinylpyridine and 4-butene pyridine in the preparation of the composite membrane II has a better capture effect on tumor cells than the separate addition of 2-vinylpyridine or the separate addition of 4-butene pyridine. This may be because the amino groups and sulfonic acid groups in 2-vinylpyridine and 4-butene pyridine react during the mixing process, which prolongs the side chain of the obtained zwitterionic polymer, further improves the structure of the modified composite membrane II, forms an attachment site for tumor cells, and improves the capture efficiency for tumor cells.

[0089] Compared with Example 2, the capture efficiency of the microfluidic chip obtained in Example 5 and Example 6 for MDA-MB-231 cells is increased by 4.66% and 5.64% respectively, the capture efficiency for A549 cells is increased by 4.03% and 4.53% respectively, and the capture efficiency for HepG2 cells is increased by 4.24% and 4.88% respectively. It can be seen that, in the process of preparing the composite membrane II, the addition of 2-vinylpyridine, 4-butenylpyridine and butyl acrylate at the same time helps to further enhance the capture effect of the obtained modified filter membrane on tumor cells, and the addition amount of butyl acrylate is positively correlated with the capture effect of the filter membrane on tumor cells within a certain range.

[0090] Table 2 Cell loss rate (%) of different tumor cells captured by the filtering microfluidic chip

[0091]

[0092] Referring to the data in Table 2, the cell loss rate of the microfluidic chip obtained in Examples 1-6 and Comparative Examples 2-3 during the reverse washing step for different tumor cells is reduced.

[0093] Among them, during the reverse washing step, the cell loss rate of the microfluidic chip obtained in Examples 1-4 for MDA-MB-231 cells is reduced by 8.3%, 13.9%, 14.8% and 16.7% respectively compared with Comparative Example 1; the cell loss rate of the microfluidic chip obtained in Examples 1-4 for A549 cells is reduced by 7.1%, 11.6%, 15.2% and 17.0% respectively compared with Comparative Example 1; the cell loss rate of the microfluidic chip obtained in Examples 1-4 for HepG2 cells is reduced by 7.3%, 11.9%, 14.7% and 17.4% respectively compared with Comparative Example 1. It can be seen that, compared with the base membrane, the microfluidic chip assembled by the modified porous filter membrane can greatly reduce the cell loss rate, that is, the adhesion between the tumor cells and the filter membrane is low during the separation and enrichment process of the tumor cells by the microfluidic chip assembled by the modified porous filter membrane, and the tumor cells are easy to separate and the damage to the cells is small.

[0094] This may be because, in the preparation of the composite membrane II, allyltrimethylammonium chloride and 2-acrylamido-2-methylpropanesulfonic acid groups are introduced on the surface of the base membrane, the hydrophilicity of the filter membrane is enhanced, and a layer of water film is formed on the surface of the filter membrane during sampling, thereby reducing the interaction force between the tumor cells and the filter membrane, so that the tumor cells are more easily detached during the reverse washing process and the damage is small. Moreover, the surface of the modified porous filter membrane is a grafted zwitterionic polymer layer, and the interaction force between the tumor cells and the zwitterionic polymer layer is mainly due to the interaction between the positive and negative atoms in the zwitterionic polymer and the cell surface, and the interaction force is small, so the tumor cells are easy to separate.

[0095] In addition, compared with Example 1, the cell loss rates of the microfluidic chips obtained in Examples 2-3 for the three tumor cells in the reverse flushing step are all greatly reduced, which shows that the addition of 2-vinylpyridine and 4-butene pyridine in the preparation of the composite film II can reduce the cell loss rate. This may be because the addition of 2-vinylpyridine and 4-butene pyridine introduces pyridine groups in the synthesis of the composite film II, which may help to further reduce the force of the positive and negative atoms in the zwitterion on the cell surface.

[0096] In addition, the cell loss rates of the microfluidic chips obtained in Comparative Examples 2-3 for MDA-MB-231 cells, A549 cells, and HepG2 cells in the reverse flushing step are all lower than that of Comparative Example 1, but higher than that of Example 2. This shows that the addition of 2-vinylpyridine and 4-butene pyridine in the preparation of the composite film II can improve the protection effect of the modified porous filter membrane on tumor cells, compared with the addition of 2-vinylpyridine or 4-butene pyridine alone. This may be because the simultaneous presence of amino groups and sulfonic acid groups helps to improve the uniformity of the water film attached to the surface of the composite film II.

[0097] Compared with Example 2, the cell loss rates of the microfluidic chips obtained in Examples 5 and 6 for MDA-MB-231 cells in the reverse flushing step are reduced by 8.6% and 9.7% respectively, for A549 cells are reduced by 8.1% and 10.1% respectively, and for HepG2 cells are reduced by 8.3% and 12.5% respectively. This shows that the simultaneous addition of 2-vinylpyridine, 4-butene pyridine, and butyl acrylate in the preparation of the composite film II helps to further reduce the adhesion between tumor cells and the obtained modified filter membrane.

[0098] The conventional operations in the operation steps of the present application are well known to those skilled in the art, and will not be described here.

[0099] The above-described examples have described the technical solutions of the present application in detail, and it should be understood that the above-described examples are only specific embodiments of the present application and are not intended to limit the present application. Any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A filtering microfluidic chip for separating circulating tumor cells, comprising a cover plate and a bottom plate, characterized in that, the cover plate and the bottom plate are separable structures, and the opposite sides of the cover plate and the bottom plate are bonded with PDMS, and the PDMS is provided with a porous filter membrane area; a passage is arranged between the cover plate and the bottom plate, and the passage is communicated with the porous filter membrane area; the porous filter membrane area is configured with a modified porous filter membrane, and the modified porous filter membrane is a composite membrane with a three-layer structure; including a base film layer, a membrane intermediate layer and a zwitterionic polymer layer arranged in turn from inside to outside, wherein the zwitterionic polymer layer has a pyridine group; the preparation steps of the membrane intermediate layer include: immersing a clean and dry base film in a polyethyleneimine mixed solution, reacting for 6-24h, to obtain a composite membrane I with a membrane intermediate layer; the polyethyleneimine mixed solution includes polyethyleneimine and Tris-HCl buffer solution, and also contains 3-phthaloylimidopropionaldehyde; the mass ratio of polyethyleneimine to 3-phthaloylimidopropionaldehyde is 1:0.5-2.

2. The filtering microfluidic chip for separating circulating tumor cells according to claim 1, characterized in that, the pore size of the modified porous filter membrane is 5-15μm.

3. A modified porous filtration membrane, characterized by, including a base film layer, a membrane intermediate layer and a zwitterionic polymer layer arranged in turn from inside to outside; the zwitterionic polymer layer has a pyridine group, and the pyridine group is derived from 2-vinylpyridine and / or 4-butene pyridine; the preparation steps of the membrane intermediate layer include: immersing a clean and dry base film in a polyethyleneimine mixed solution, reacting for 6-24h, to obtain a composite membrane I with a membrane intermediate layer; the polyethyleneimine mixed solution includes polyethyleneimine and Tris-HCl buffer solution, and also contains 3-phthaloylimidopropionaldehyde; the mass ratio of polyethyleneimine to 3-phthaloylimidopropionaldehyde is 1:0.5-2.

4. A method for producing a modified porous filter membrane, characterized by, including the following steps: S1. Preparation of a membrane intermediate layer: immerse a clean and dry base film in a polyethyleneimine mixed solution, react for 6-24h, to obtain a composite membrane I with a membrane intermediate layer; S2. Preparation of a grafted modified microfiltration membrane: treat the composite membrane I in S1 with an oxidation-reduction initiator, and react with a zwitterionic polymer monomer and 2-vinylpyridine and 4-butene pyridine to obtain a base film with a grafted modified microfiltration membrane, denoted as a composite membrane II; In step S1, the polyethyleneimine mixed solution includes polyethyleneimine and Tris-HCl buffer solution, and also contains 3-phthaloylimidopropionaldehyde; the mass ratio of polyethyleneimine to 3-phthaloylimidopropionaldehyde is 1:0.5-2.

5. The preparation method of the modified porous filter membrane according to claim 4, characterized in that, in step S2, the zwitterionic polymer monomer is added before 2-vinylpyridine and 4-butene pyridine. The zwitterionic polymer monomer includes at least one of allyl trimethyl ammonium chloride, 2-acrylamido-2-methylpropanesulfonic acid, and polycarboxybetaine, and a mass concentration of the zwitterionic polymer monomer is 3-10 mg / mL before 2-vinylpyridine and 4-butenylpyridine are added. 6.The method of claim 4, wherein the step of modifying the porous filter membrane is performed by adding a zwitterionic polymer monomer to the porous filter membrane. In step S2, the volume ratio of 2-vinylpyridine to 4-butenylpyridine is 1-2:1-3. 7.The method of claim 4, wherein the step of modifying the porous filter membrane is performed by adding a zwitterionic polymer monomer to the porous filter membrane. In step S2, the mass volume ratio of the zwitterionic polymer monomer to 2-vinylpyridine and 4-butenylpyridine is 3-6 mg:0.5-2.5 mL:0.5-2.5 mL.

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